CN110754471A

CN110754471A - Insecticidal activity of AMEP412 protein on trialeurodes vaporariorum and application of insecticidal activity to trialeurodes vaporariorum

Info

Publication number: CN110754471A
Application number: CN201911212945.9A
Authority: CN
Inventors: 刘权; 肖莉杰; 殷奎德
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2020-02-07
Anticipated expiration: 2039-12-02
Also published as: NL2026309A; CN110754471B; NL2026309B1

Abstract

The invention relates to application of bacillus subtilis protein AMEP412 in controlling trialeurodes vaporariorum. The invention discovers the insecticidal activity of the AMEP412 protein on trialeurodes vaporariorum and the application thereof in controlling the trialeurodes vaporariorum, the protein can kill the trialeurodes vaporariorum in low concentration, has the stability of heat resistance and natural degradation resistance, can be taken into intestinal tracts by the trialeurodes vaporariorum, and does not inhibit the activity of protease in the intestinal tracts; in addition, the AMEP412 sprayed in the greenhouse has good control effect on the trialeurodes vaporariorum; the invention accumulates new materials for the biological control work of the trialeurodes vaporariorum.

Description

Insecticidal activity of AMEP412 protein on trialeurodes vaporariorum and application of insecticidal activity to trialeurodes vaporariorum

Technical Field

The invention belongs to the field of plant protection and biological control, and relates to insecticidal activity of AMEP412 protein on trialeurodes vaporariorum and application of the insecticidal activity to the trialeurodes vaporariorum.

Background

Whitefly (Bemisia tabaci) is an important pest of various crops such as vegetables, cotton, ornamental plants, and the like. It damages crops by eating plant stem and leaf juice and spreading plant viruses, resulting in huge losses in agricultural production and national economy. The main strategy for preventing and treating powdery mildew is mainly chemical insecticide. However, whiteflies develop resistance to those insecticides which are used excessively and frequently. In view of the above, the search for novel insecticides should become a new research focus.

Compared with chemical pesticides, proteins with insecticidal activity have relatively low probability of resisting insects due to different action modes, and become a hotspot of current pesticide research. There have been many reports on the use of pesticidal proteins in pest control. The best known example is the Cry toxin of Bacillus thuringiensis (Bt), which was developed into Bt transgenic cotton and effectively controls lepidopteran pest larvae. The wide planting of the Bt transgenic cotton greatly reduces the use of chemical pesticides. However, Cry toxins are not effective against trialeurodes vaporariorum. In recent years, some researchers have focused on screening for insecticidal proteins from plants that are rarely infested with trialeurodes vaporariorum. Das et al, 2009, reported that a mannose-binding lectin, extracted from garlic leaves, was effective in inhibiting whitefly. Jin et al expressed the lectin of pinellia ternata in chloroplasts in 2012 and demonstrated its ability to resist trialeurodes vaporariorum. Shukla et al identified a pesticidal protein (Tma12) from an edible fern in 2016 and expressed in transgenic cotton, which protein exhibited some resistance to white whitefly. Although these insecticidal proteins from plants have some potential for controlling whitefly, they are not widely used at present due to low extraction rates and limited application of transgenes.

In previous researches, the protein AMEP412 is separated and identified from the bacillus subtilis BU412, can stimulate defense response of plants, improve disease resistance of the plants, can generate antagonism on streptomyces scabies, and is an ideal candidate for developing biological pesticides. In the present invention, we aimed to provide a novel use of the AMEP412 protein.

Disclosure of Invention

The invention aims to provide a new application of bacillus subtilis protein AMEP412, which solves the problem that the existing insecticidal protein for trialeurodes vaporariorum is too little and solves the problem of green prevention and control of the trialeurodes vaporariorum.

The invention is realized by the following technical scheme: an application of Bacillus subtilis protein AMEP412 in controlling whitefly.

The amino acid sequence of the Bacillus subtilis protein AMEP412 is shown in SEQ ID NO. 1.

Furthermore, after the AMEP412 protein is fed to whitefly adults, the protein concentration required for achieving half lethal dose is 15.57 mu g/ml, and the protein concentration required for achieving 90% lethal rate is 63.27 mu g/ml.

Further, the AMEP412 protein can be ingested by the whitefly adults into the body, and is intensively positioned in the intestinal tract.

Further, the AMEP412 protein has heat-resistant stability.

Further, the AMEP412 protein has stability to natural degradation.

Further, the AMEP412 protein is sprayed in a greenhouse at a concentration of 100 mu g/ml, and the fatality rate of trialeurodes vaporariorum after 2 days is 56%.

Firstly, AMEP412 protein with different concentration gradients is used for artificially feeding the white fly imagoes, and the half fatality rate (LC) is calculated₅₀) And protein concentration required for 90% lethality. And then carrying out heat treatment and natural degradation treatment on the AMEP412 protein, detecting the insecticidal activity of the treated protein sample on the white whitefly imagoes, and determining the stability of the AMEP412 protein. And (3) marking AMEP412 protein by using FITC, observing the fluorescence distribution in the trialeurodes vaporariorum by using a fluorescence microscope after feeding the adult trialeurodes vaporariorum, and determining the location of the AMEP412 in the trialeurodes vaporariorum. The inhibition of trypsin by AMEP412 was tested, excluding the possibility of killing whitefly by inhibiting its intestinal protease activity.

Adopt above-mentioned technical scheme's positive effect: the invention discovers the insecticidal activity of the AMEP412 protein on trialeurodes vaporariorum and the application thereof in controlling the trialeurodes vaporariorum, the protein can kill the trialeurodes vaporariorum in low concentration, has the stability of heat resistance and natural degradation resistance, can be taken into intestinal tracts by the trialeurodes vaporariorum, and does not inhibit the activity of protease in the intestinal tracts; in addition, the AMEP412 sprayed in the greenhouse has good control effect on the trialeurodes vaporariorum; the invention accumulates new materials for the biological control work of the trialeurodes vaporariorum.

Drawings

Fig. 1 is a graph of mortality of varying concentrations of the AMEP412 protein to whitefly adults, with the same letters indicating no significant difference and different letters indicating significant differences between treatments (α -5%);

fig. 2 is a graph showing the lethality of heat-treated and naturally degraded AMEP412 protein to whitefly adults, CK indicates that no AMEP412 protein was added, and unored indicates that the AMEP412 protein was not treated, the same letters indicate no significant difference, and different letters indicate significant difference between treatments (α ═ 5%);

fig. 3 is a fluorescent localization of FITC-labeled AMEP412 in whitefly adults, with bright spots in the abdominal cavity as excited green fluorescence, showing that AMEP412 is localized in the gut.

Detailed Description

The present invention is further described below by way of examples, it being understood that these examples are for illustrative purposes only and do not limit the scope of the present invention in any way.

Sources of the biological material in the present invention:

1. the used Bacillus subtilis BU412 is preserved in the China center for type culture Collection in 2016, 3, 30 days, with the preservation number of CCTCC M2016142;

2. AMEP412 protein: patent application No. 201810928176.1 entitled "identification of a novel function of Bacillus subtilis AMEP412 protein and its antibacterial peptide", application date 20180814, publication date 20181228, publication No. CN 109096379A.

Example 1

This example illustrates the insecticidal effect of Bacillus subtilis AMEP412 protein when artificially fed to Bemisia alba.

Liquid artificial diet for feeding was sterilized after 5% yeast extract and 30% sucrose were dissolved in distilled water, AMEP412(1, 5, 10, 20, 40 and 80. mu.g/ml) was added at various concentrations, mixed for use, AMEP412 was used as a control, adult whitefly adults at 1-2 days old were expelled from plant leaves into 50ml test tubes, at least 50 adult whitefly were taken in each test tube, two layers of stretched sealing film were covered on the tube cap of the test tube, bait (100. mu.l) was added between the two layers of film, then the tube cap was covered reversely over the tube cap so that the double-layered film structure was maintained inside the test tube and was placed vertically upward, mortality was measured after feeding three times for each set of experiments, mortality was recorded by counting dead adult whitefly adults at the bottom of the test tube, data was analyzed by single factor analysis (ANOVA), and lethality concentration was calculated by SPSS 50% lethal concentration analysis (SPSS 32% lethal concentration) using Tukey's-HSD test (α ═ 5%).

The mortality rate of trialeurodes vaporariorum was counted after 2 days of feeding as shown in fig. 1. With the increase of the AMEP412 protein concentration, the mortality rate of the trialeurodes vaporariorum is gradually increased and shows a positive correlation trend. The mortality rates of trialeurodes vaporariorum corresponding to the AMEP412 protein concentrations of 0, 1, 5, 10, 20, 40, 80mg/ml were 7.2%, 17.4%, 31.0%, 47.9%, 65.5%, 82.7% and 95.6%, respectively. The software calculated the AMEP412 protein concentration required to achieve half-lethal rate was 15.57. mu.g/ml (Table 1). In addition, the AMEP412 protein concentration required to achieve 90% lethality was 63.27 μ g/ml. These results indicate that the AMEP412 protein has insecticidal activity against the whitefly adults, and the required half-lethal concentration is low, indicating that the insecticidal activity is strong.

TABLE 1 calculation of the median lethal concentration of AMEP412 protein

Example 2

This example illustrates the stability experiments of the Bacillus subtilis AMEP412 protein.

To test the heat resistance of the AMEP412 protein, the protein samples were treated at 95 ℃ for 15 minutes and 30 minutes, respectively, the cooled protein samples were prepared as artificial baits according to the method of example 1, and the final concentration was adjusted to 60. mu.g/ml for insecticidal effect test of Bemisia alba, the untreated protein samples were used as controls, each treatment was repeated three times, in order to test the stability of the AMEP412 protein against natural degradation, the protein samples were placed in 1.5ml centrifuge tubes and left to stand at room temperature of 25 ℃ for 24 hours and 48 hours, respectively, after which the protein samples were prepared as artificial baits according to the method of example 1, the final concentration was adjusted to 60. mu.g/ml for insecticidal effect test of Bemisia alba, the untreated protein samples were used as controls, each treatment was repeated three times, and the mortality data of Bemisia alba were compared using one-way analysis of variance with Tukey's HSD test (α ═ 5%).

The results show (fig. 2) that the mortality of aleurodes vaporariorum is reduced by 0.57% and 0.49% respectively by the treatment of the AMEP412 protein at 95 ℃ for 15 minutes and 30 minutes, compared with the AMEP412 protein without heat treatment, and the difference is not significant. Indicating that AMEP412 has good thermal stability. Compared with the AMEP412 protein which is not subjected to natural placement treatment, the AMEP412 protein reduces the fatality rate of the white whitefly by 2.73% after being naturally placed for 24 hours at 25 ℃, and the difference is not obvious. And after the AMEP412 protein is naturally placed at 25 ℃ for 48 hours, the fatality rate to the trialeurodes vaporariorum is reduced by 16.38%. Although a significant difference was achieved, the mortality rate was still 70%, so the AMEP412 protein was considered to have good stability to natural degradation. These results demonstrate that AMEP412 has stability to heat and to natural degradation, facilitates application in high temperature environments, and prolongs its duration of action.

Example 3

This example illustrates the fluorescent localization of the Bacillus subtilis AMEP412 protein in whitefly.

And (3) incubating the AMEP412 protein with fluorescein FITC, and removing FITC which is not combined with the AMEP412 protein according to the molecular volume difference by molecular sieve chromatography to obtain the FITC-labeled AMEP412 protein. FITC-labeled AMEP412 protein was subsequently fed to adult whitefly. Dead whitefly adults were washed with distilled water and then fluorescence microscopic observation was performed by exciting green fluorescence at a wavelength of 495nm using an Olympus BX60 fluorescence microscope. The results show (fig. 3) that the green fluorescence is concentrated in the intestinal tract of the adult whitefly, indicating that the AMEP412 protein can be taken up by the adult whitefly into the body and localized in the intestinal tract. This result suggests that our AMEP412 is in the gut where it exerts its insecticidal effect.

Example 4

This example illustrates the effect of Bacillus subtilis AMEP412 protein on trypsin activity.

Some insecticidal proteins act by acting as protease inhibitors, by inhibiting the activity of serine proteases (e.g., trypsin) in the insect gut, which render the insect indigestible and the ingested food lethal. To determine whether the Bacillus subtilis AMEP412 protein acts as a protease inhibitor to achieve pesticidal effects, this example examined the effect of AMEP412 protein on trypsin activity.

The trypsin activity test is based on the detection of the product catalyzed by trypsin on the substrate N- α -benzoyl-DL-arginine-p-nitroanilide (BAPNA) with a specific absorption at 405nm the effect on the activity of trypsin can be determined by measuring the change in absorbance 0.2ml of trypsin solution (0.1mg/ml), 0.2ml of AMEP412 protein (0.1mg/ml) and 1.6ml of buffer (0.2M Tris HCl, pH7.8) are incubated in a reaction cup for 3 minutes the enzymatic reaction is started by adding 1ml of substrate (1mg/ml BAPNA) and the solution is mixed immediately, recording the increase in absorbance at 405nm for 5 minutes, with no AMEP protein as control, the inhibition is repeated three times per treatment.100% for 405nm minutes: (AMEP412 treatment. DELTA. 405 nm/min-control DELTA.A 405/405 nm/min): 100/nm min).

The results show (table 2) that the inhibition rate of the AMEP412 protein on trypsin is 2.97%, and there is no significant difference. The amap 412 protein is shown to have no inhibitory effect on trypsin activity, suggesting that the insecticidal activity of the AMEP412 is not a mechanism of action of protease inhibitors.

TABLE 2 Effect of AMEP412 protein on Trypsin Activity

Example 5

This example illustrates the control effect of Bacillus subtilis AMEP412 protein on whitefly in the greenhouse.

Tomato plants were grown in a greenhouse to a height of about 50cm, and three tomatoes were treated as one, covered with a gauze, and inoculated with at least 200 adult whiteflies of 1-2 days old per gauze. The purified exciton AMEP412 protein sample was diluted to 100. mu.g/ml for spray treatment, with buffer as control, and each treatment was repeated 3 times. And (5) counting the mortality of the trialeurodes vaporariorum 2 days after spraying.

The results show (table 3), the mortality of the trialeurodes vaporariorum is 56.2% after 100 mug/ml of AMEP412 protein is sprayed for two days, and has a very significant difference (α is 1%), although the mortality is reduced compared with feeding experiments, the inhibition rate of the trialeurodes vaporariorum can reach more than 50% by considering the influence of various factors in the actual environment, so the bacillus subtilis AMEP412 protein has a good prevention and control effect on the trialeurodes vaporariorum in a greenhouse.

TABLE 3 Effect of AMEP412 protein on controlling Bemisia alba in greenhouse

The invention discovers the insecticidal activity of the bacillus subtilis protein AMEP412 on whitefly imagoes, further confirms the stability of the protein on heat treatment and natural degradation, detects the positioning of the protein in whitefly bodies, and confirms the cracking effect of the protein on insect cells. Furthermore, the invention detects the control effect of the protein on the trialeurodes vaporariorum in a greenhouse, and accumulates new materials and experiences for biological control work.

Sequence listing

<110> university of eight agricultural reclamation of Heilongjiang

<120> insecticidal activity of AMEP412 protein on trialeurodes vaporariorum and application thereof in control of trialeurodes vaporariorum

<141>2019-12-02

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<170>SIPOSequenceListing 1.0

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Pro Trp Gly Lys Val Ala Ser Phe Leu Lys Trp Ala Gly Asn Leu Ala

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Ala Ala Ala Ala Lys Tyr Ser Tyr Thr Ser Gly Lys Lys Ile Leu Ala

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Tyr Ile Gln Lys His Pro Gly Lys Ile Val Asp Trp Phe Leu Lys Gly

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Tyr Ser Val Tyr Asp Val Ile Lys Met Ile Leu Gly

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Claims

1. An application of Bacillus subtilis protein AMEP412 in controlling whitefly.

2. The Bacillus subtilis protein AMEP412 of claim 1, which has the amino acid sequence shown in SEQ ID NO 1.

3. Use according to claim 1, characterized in that: after the AMEP412 protein is fed to whitefly adults, the protein concentration required for achieving half lethal dose is 15.57 mu g/ml, and the protein concentration required for achieving 90% lethal rate is 63.27 mu g/ml.

4. Use according to claim 1, characterized in that: the AMEP412 protein can be ingested into the body by whitefly adults and is concentrated in the intestinal tract.

5. Use according to claim 1, characterized in that: the AMEP412 protein has heat-resistant stability.

6. Use according to claim 1, characterized in that: the AMEP412 protein has stability to natural degradation.

7. Use according to claim 1, characterized in that: the AMEP412 protein is sprayed in a greenhouse at a concentration of 100 mu g/ml, and the mortality of trialeurodes vaporariorum after 2 days is 56%.